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Abstract
Rhabdomyosarcomas (RMS) are a heterogeneous group of mesodermal tumors, the most common sub-types are embryonal (eRMS) and alveolar (aRMS) rhabdomyosarcoma. Immunohistochemical analysis revealed c-Myb expression in both eRMS and aRMS. c-Myb has been reported to be often associated with malignant human cancers. We therefore investigated the c-Myb role in RMS using cellular models of RMS. Specific suppression of c-Myb by a lentiviral vector expressing doxycycline (Dox)-inducible c-Myb shRNA inhibited proliferation, colony formation, and migration of the eRMS cell line (RD), but not of the aRMS cell line (RH30). Upon c-Myb knockdown in eRMS cells, cells accumulated in G0/G1 phase, the invasive behaviour of cells was repressed, and elevated levels of myosin heavy chain, marker of muscle differentiation, was detected. Next, we used an RD-based xenograft model to investigate the role of c-Myb in eRMS tumorigenesis in vivo. We found that Dox administration did not result in efficient suppression of c-Myb in growing tumors. However, when c-Myb-deficient RD cells were implanted into SCID mice, we observed inefficient tumor grafting and attenuation of tumor growth during the initial stages of tumor expansion. The presented study suggests that c-Myb could be a therapeutic target in embryonal rhabdomyosarcoma assuming that its expression is ablated.
Highlights
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma[1]
To investigate whether c-Myb plays a role in RMS tumorigenesis, we assessed the effects of c-Myb suppression in embryonal (RD) and alveolar (RH30) RMS cell lines[27]
In the RD cell line, Dox induction (5 μg/ml)[28] of Myb shRNA abolished c-Myb expression, but the c-Myb levels were not affected in cell transduced with empty pLVTSH (Fig. 1a)
Summary
Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma[1] These sarcomas represent a heterogeneous group of malignancies that express myogenic regulatory transcription factors such as MyoD, myogenin[2] and myf[53,4] and exhibit defective skeletal muscle differentiation[5]. (a) Western blot shows c-Myb expression in RD cells lentivirally transduced with a Dox - inducible c-Myb shRNA (RDshMYB) vector or empty vector (RDEmpty) 48 hours after Dox induction (5 μg/ml). Western blot shows c-Myb expression in RH30 cell lentivirally transduced with a Dox-inducible c-Myb shRNA (RH30shMYB) vector and treated with Dox at 1 and 2.5 μg /ml. Comparison of the effect of c-Myb silencing on RD (f) and RH30 (g) cell line proliferation after six days of treatment with or without Dox as measured by crystal violet staining. Cells were grown with or without Dox, as indicated, (Dox at 5 μg/ml) for four days and analysed by propidium staining and flow cytometry
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